Modification of plant development and morphology

ABSTRACT

A method of modifying morphology in a plant comprising introducing into a plant at least one chimaeric gene comprising a promoter sequence operably associated with a nucleic acid sequence, the promoter sequence being operable to direct expression in specific cells of the plant and the nucleic acid sequence encoding at least one gene product capable of altering the metabolism of or causing death of the specific cells and/or nearby cells. In particular, the promoter sequence is operable to direct expression in lateral bud or lateral shoot and the nucleic acid encoding at least one gene product capable of disrupting the metabolism of or causing the death of the lateral bud or lateral shoot or nearby cells. Preferably the promoter sequence comprises the sequence shown as SEQ ID No. 1 or SEQ ID No. 7 or SEQ ID No. 4, or a part thereof capable of regulating expression of a gene, or a sequence having at least 60%, preferably at least 75%, homology to SEQ ID No. 1 or SEQ ID No. 7 and being capable of regulating expression of a gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority under 35U.S.C. §120 to U.S. Non-Provisional patent application Ser. No.13/160,239, filed Jun. 14, 2011, now U.S. Pat. No. 8,575,423 issued Nov.5, 2013 and entitled “Modification of Plant Development and Morphology,”which is a continuation of and claims priority under 35 U.S.C. §120 toU.S. Non-Provisional patent application Ser. No. 11/729,514, filed Mar.28, 2007, now U.S. Pat. No. 8,093,459 issued Jan. 10, 2012 and entitled“Modification of Plant Development and Morphology,” which is acontinuation of international PCT application Serial No.PCT/GB2005/003719 filed Sep. 28, 2005 and published as WO2006/035221 inEnglish on Apr. 6, 2006, which claims priority under 35 U.S.C. §119 toGreat Britain Application 0421598.4 filed on Sep. 29, 2004. The entirecontents of the aforementioned applications are herein expresslyincorporated by reference.

This application hereby incorporates by reference the sequence listingin the text file, named “BTMK-024_02US_318520-2360_TEXT.txt,” filedherewithin and having a size of 15 KB.

FIELD OF THE INVENTION

The invention relates to a method for modifying the development andmorphology of a plant. The invention further relates to the use of a DNAsequence to regulate exogenous gene expression in tissues of a plant.The invention yet further relates to an identified DNA sequence whichact as an promoter which is operable to direct expression in specificcells of a plant.

BACKGROUND

The control of plant morphology is of major importance in the commercialproduction of plants for agricultural or horticultural purposes, toenhance productivity and yield, to improve the efficiency of husbandryand harvest, and to achieve aesthetic desirability. Features whichrequire control or modification may include the morphology of theflower, fruit or tuber, the quantity of flowers, fruit, seed or tubers,the extent of primary and lateral roots, the form of the aerial shootsor trunk, and the presence of thorns or stinging hairs. Other featureswhich may be desirably controlled include the advancement or delay ofabscission of leaves, flowers or fruit, the release of seeds, and theproduction of storage organs or secretory glands.

Morphological changes often occur as a result of environmental impact onthe plant, including physical damage, herbivore predation, pathogeninfection, cold, heat, and drought. They can often be brought aboutdeliberately by human intervention, either physically (pruning, bending,tying, staking, or excising particular organs or structures) orchemically (application of agrochemicals and plant growth substances).Whichever is the causative agent, morphological changes are enacted byexpression of genes within the cells of the plant itself. At the onsetof the change, the initiation of expression of one or more genes occursin those particular tissues where cell growth, proliferation,development or necrosis is required to culminate in the gross physicalchange.

The expression of a gene is dependent upon its DNA sequence beingtranscribed into RNA by the action of RNA polymerase. To achieve this,RNA polymerase must recognise and attach to a region of DNA sequencelocated upstream of (i.e. 5′ to) the gene coding sequence in order fortranscription to be initiated. Such a region is termed the promoter ofthe gene. The intrinsic nature of the promoter sequence determines thecircumstances and the manner in which the gene is expressed.

There are, broadly speaking, four types of promoters found in planttissues; constitutive, tissue-specific, developmentally-regulated, andinducible/repressible, although it should be understood that these typesare not necessarily mutually exclusive.

A constitutive promoter directs the expression of a gene throughout thevarious parts of a plant continuously during plant development, althoughthe gene may not be expressed at the same level in all cell types.Examples of known constitutive promoters include those associated withthe cauliflower mosaic virus 35S transcript (Odell et al, 1985), therice actin 1 gene (Zhang et al, 1991) and the maize ubiquitin 1 gene(Cornejo et al, 1993).

A tissue-specific promoter is one which directs the expression of a genein one (or a few) parts of a plant, usually throughout the lifetime ofthose plant parts. The category of tissue-specific promoter commonlyalso includes promoters whose specificity is not absolute, i.e. they mayalso direct expression at a lower level in tissues other than thepreferred tissue. Examples of tissue-specific promoters known in the artinclude those associated with the patatin gene expressed in potato tuberand the high molecular weight glutenin gene expressed in wheat, barleyor maize endosperm.

A developmentally-regulated promoter directs a change in the expressionof a gene in one or more parts of a plant at a specific time duringplant development. The gene may be expressed in that plant part at othertimes at a different (usually lower) level, and may also be expressed inother plant parts.

An inducible promoter is capable of directing the expression of a genein response to an inducer. In the absence of the inducer the gene willnot be expressed. The inducer may act directly upon the promotersequence, or may act by counteracting the effect of a repressormolecule. The inducer may be a chemical agent such as a metabolite, aprotein, a growth regulator, or a toxic element, a physiological stresssuch as heat, wounding, or osmotic pressure, or an indirect consequenceof the action of a pathogen or pest. A developmentally-regulatedpromoter might be described as a specific type of inducible promoterresponding to an endogenous inducer produced by the plant or to anenvironmental stimulus at a particular point in the life cycle of theplant. Examples of known inducible promoters include those associatedwith wound response, such as described by Warner et at (1993),temperature response as disclosed by Benfey & Chua (1989), andchemically induced, as described by Gatz (1995).

A promoter sequence may comprise a number of defined domains necessaryfor its function. A first of these comprises approximately 70 base pairslocated immediately upstream of (that is, 5′ to) the structural gene andforms the core promoter. The core promoter contains the CAAT and TATAboxes and defines the transcription initiation site for the gene. Aseries of regulatory sequences upstream of the core promoter constitutethe remainder of the promoter sequence and determine the expressionlevels, the spatial and temporal patterns of expression, and theresponse to inducers. In addition some promoters contain sequenceelements which act to enhance the level of expression, for example thatfrom the pea plastocyanin promoter as described in International PatentPublication No. WO 97/20056.

Genetic modification of plants depends upon the introduction ofchimaeric genes into plant cells and their controlled expression underthe direction of a promoter. Promoters may be obtained from differentsources including animals, plants, fungi, bacteria, and viruses, anddifferent promoters may work with different efficiencies in differenttissues. Promoters may also be constructed synthetically.

It may often be desirable to express introduced genes in a number ofdifferent tissues within a plant. For example the expression of aresistance to a pathogen or pest, or tolerance to temperature extremesmight be best expressed throughout all tissues in a plant. Similarly itmight be desirable to ensure the expression of the transgenes at alltimes throughout the development of the plant. Also, a promoter which isexpressed in a manner that is immune to the influence of inducers orrepressors resulting from unforeseen environmental stimuli may also beuseful to ensure the continued expression of a trait. For thesepurposes, the use of a “constitutive” promoter would be desirable.Examples of constitutive promoters include the CaMV 35S promoter. Forcereals the ubiquitin promoter is a constitutive promoter of choice(Christensen & Quail, 1996).

However, in some instances it is more desirable to control the locationof gene expression in a transgenic plant. This may enhance the effect ofgene expression by ensuring that expression occurs preferentially inthose tissues where the effect of the gene product is most efficacious.By the same argument, modulated expression can reduce potential yieldloss by limiting the resource drain on the plant. Further advantagesinclude limiting the expression of agronomically useful yet generallydeleterious genes to specific tissues by localisation andcompartmentalisation of gene expression in cases where the gene productmust be restricted to, or excluded from, certain tissues. For example,anther specific expression of the suc inhibitor genes (Mariani et al,1990) has been used in male sterility systems, whereas expression inother parts of the plant would result in toxicity. A similar cell deathsystem is described in International Patent Application WO 89/10396where an RNAse protein is used in combination with an anther specificpromoter to cause necrosis of the anther cells and confer male sterilityon the plant.

In International Patent Applications WO 02/33106 and WO 02/33107 aredescribed plant cell death systems providing resistance to nematodeinfection by the expression of a ribosome inactivating protein (MaizeRibosome Inactivating Protein, Pokeweed Antiviral Protein) under theregulation of nematode feeding site specific promoters. In these casesthe specificity of expression of the deleterious gene is enhanced by thepromoters being both tissue specific and responsive to nematodeinvasion.

In some instances two or more transgenes may be expressed in a plant insimilar or different locations. Each transgene may be expressed underthe control of a different promoter which expresses in more than oneregion of the plant. The promoters may be selected so that there is anoverlap in their respective expression sites at one or more desiredlocations. This overlap site(s) gives increased specificity andtargeting of gene expression. By judicious selection of the gene productencoded by each transgene, the overlapping expressing of both transgenesmay lead to an additive or enhanced effect on the target tissues,whereas expression of only one or other of the transgenes at otherlocations may cause no effect on the plant. For example in InternationalPatent Application WO 02/33106 two separate peptide domains derived fromMaize Ribosomal Inhibitory Protein (RIP) are expressed under theregulation of two different tissue specific promoters, having differentexpression profiles but which nevertheless have one site in common,resulting in the production of an active protein at the site of overlap.

Conversely, the two transgenes may encode an effector molecule and anagonist molecule or protectant. In this case, the effector molecule willaffect the plant at all locations where it is expressed, except thosewhere the expression site overlaps with that of expression of theagonist or protectant molecule. In NZ 260511 a plant cell death systemis proposed with increased tissue specificity. This system comprises theexpression of a cytotoxic molecule (under the control of a firstpromoter, which first promoter causes expression in specific targetcells and at one or more other sites in the plant), in conjunction witha protective molecule (under the control of a second promoter, whichsecond promoter causes expression in all of the sites where the firstpromoter is active except the specific target cells). Examples ofsuitable cytotoxic and protective molecules are proteases and proteaseinhibitors, respectively, or nucleases and nuclease inhibitors,respectively. WO 93/10251 discloses the use of a cytotoxic ribonucleasemolecule Barnase together with the protective inhibitor moleculeBarstar.

Another example of a two-component transgenic system is provided inInternational Patent Application No. WO98/44138. This system comprisesthe expression of a gene product under the control of a promoter, whichpromoter and gene product are selected so that there is an overlap intheir respective expression and effector sites at a desired location.The promoter directs expression in the specific cells and also at one ormore other sites in the plant, whilst the molecular target of the geneproduct occurs in a second range of cells also including the specifictarget cells. This overlap site(s) gives increased specificity andtargeting of gene expression to the cells at the desired location. Byjudicious selection of the gene product encoded by the transgene, theexpression in non-target cells causes no effect on the plant.

A major application of the localised expression of a deleterious gene toa particular tissue, would be in the modification of plant morphology,for example by the controlled necrosis or prevention of development ofcertain tissues or organs, such as flowering structures, fruitingbodies, storage tissues, shoots, leaf tissues, root tissues, abscissionzones, secretory glands, stinging cells, trichomes, or thorns.

A particular application of localised expression of a deleterious geneto modify plant morphology would be in the prevention of lateral shootoutgrowths from leaf axillary meristems. The anatomy of axillarymeristems and lateral buds is described in Esau (1960). Outgrowth oflateral shoots most commonly arises when the dominance of the apicalshoot is removed or reduced; for example when the apical shoot isdamaged or removed, either accidentally through physical damage orpredation by herbivores, or as part of agricultural practice e.g.coppicing. Other changes which modify for example the production,transport, detection, or metabolism of endogenous plant growthsubstances may also cause outgrowth from axillary meristems. Lateralshoots, or “suckers”, may be undesirable for purely aesthetic reasons,may produce a plant with unusable morphology, or may have a detrimentalmetabolic effect on the plant as a whole by acting as an additionalsource or sink for various metabolites or plant growth substances.

One example where lateral bud outgrowth occurs is in the commercialcultivation of tobacco, where the apical shoot comprising theinflorescence and uppermost leaves is removed at a specific time duringthe growth of the plant, in a process named “topping”, to stimulategrowth and development of the remaining leaves, to enhance root growth,and to encourage the redistribution of metabolites and secondarycompounds to the plant leaves. A drawback to the topping process is thatit also stimulates the outgrowth of lateral shoots which thereby offsetsthe desired redistribution of metabolites. This effect is commonlyovercome by the physical removal of the lateral shoots which is highlylabour intensive or by the application of chemical shoot suppressantssuch as maleic hydrazide, which is both costly in terms of materials andmay result in the retention of chemical residues on the harvested plant.A system which prevents such “suckering” by specifically directing thedisruption of those cells involved in lateral bud outgrowth, wouldtherefore provide a great benefit to the cultivation of tobacco.

Ribosome inactivating proteins (RIPs) are a group of toxic plantproteins that catalytically inactivate eukaryotic ribosomes (Stirpe andBarbieri 1986). RIPs function as N-glycosidases to remove a specificadenine in a conserved loop of the large rRNA, and thereby preventbinding of Elongation Factor 2, thus blocking cellular proteinsynthesis. Three forms of RIPs have been described. Type 1 RIPs such aspokeweed antiviral protein and barley translation inhibitor are eachcomprised of a single polypeptide chain, each with an approximate M_(r)value of 30,000. Type 2 RIPs such as ricin, abrin and modeccin eachcomprise two polypeptide chains, one with RIP activity linked by adisulphide bond to the other galactose-binding lectin chain. Type 3 RIPssuch as maize RIP comprise a single polypeptide chain which subsequentlyundergoes proteolytic cleavage to release two active peptide domains.

Pokeweed (Phytolacca americana) produces three distinct antiviralproteins, namely PAP′, PAPII and PAP-S that appear in spring leaves,summer leaves and seeds, respectively. Amino acid similarities betweenthese three proteins have been observed. As used herein ‘PAP’ covers allthree of these antiviral proteins.

U.S. Pat. No. 6,015,940 discloses the preparation of a cDNA clone ofPAP′ prepared from spring leaves of pokeweed, and the use thereof underthe control of a constitutive promoter (either cauliflower mosaic virus35S promoter or the figwort mosaic virus 35S promoter) in the productionof transgenic tobacco and potato plants resistant to infection by theviruses PVX and PVY.

Transgenic plants containing the summer leaf form of PAP, PAP-II, havebeen described in WO 99/60843. A number of full length and truncatedPAP-II gene sequences were screened in order to identify those variantPAP-II proteins which retained antiviral activity but exhibited nophytotoxicity. Transgenic plants exhibited both antiviral and antifungalactivity.

The PAP gene is expressed in vivo in leaves initially to produce aninactive Pro-PAP protein. It is known that following translation, thePro-PAP′ protein molecule is targeted to the cell wall. At some stageduring this process the N- and C-terminal extensions of the Pro-PAP′molecule are cleaved to produce an activated PAP′ molecule (maturePAP′). In the case of PAP-S (expressed in seeds) the cellularlocalisation is not known. However, the N-terminal processed region ofPAP-S appears to have properties similar to signal sequences fortargeting.

The structure of the mature PAP-S protein, i.e. with N- and C-terminalextensions removed, may be described in terms of two separate domains,corresponding to the two domains of Type 3 RIPs, or the two polypeptidesof Type 2 RIPs, i.e. the ribosome binding domain and the catalyticdomain.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a method of modifyingmorphology in a plant comprising introducing into a plant at least onechimaeric gene comprising a promoter sequence operably associated with anucleic acid sequence, the promoter sequence being operable to directexpression in specific cells of the plant, and the nucleic acid sequenceencoding at least one gene product capable of altering the metabolism ofthe specific cells and/or nearby cells.

In one embodiment the gene product may be capable of enhancing themetabolism of or promoting vigour of the specific cells and/or nearbycells.

In another preferable embodiment, the gene product is capable ofdisrupting the metabolism of or causing death of the specific cellsand/or nearby cells.

In a further aspect the present invention provides a method of modifyingmorphology in a plant comprising introducing into a plant at least onechimaeric gene comprising a promoter sequence operably associated with anucleic acid sequence, the promoter sequence being operable to directexpression substantially specifically in a lateral bud and/or lateralshoot, preferably a lateral bud initiation cell, and the nucleic acidencoding at least one gene product capable of altering the metabolism ofthe lateral bud and/or lateral bud initiation cells and/or nearby cells.

In one embodiment the gene product may be capable of enhancing themetabolism of or promoting vigour of the specific cells and/or nearbycells.

In another preferred embodiment, the gene product is capable ofdisrupting the metabolism of or causing death of the specific cellsand/or nearby cells.

The term “substantially specifically” as used herein means that thepromoter according to the present invention is operable to directexpression predominantly in the lateral bud and/or lateral shoot. Thepromoter according to the present invention may in addition to beingoperable to direct expression in the lateral bud and/or lateral shootmay also be operable to direct expression in other cell type(s) ortissue(s) within the plant provided that the predominant expression(i.e. greater than at least 51% of the overall (total) expression in theplant) occurs within the lateral bud and/or lateral shoot.

The promoter according to the present invention may in addition to beingoperable to direct expression in the lateral bud and/or lateral shootmay also be operable to direct expression in other cell type(s) ortissue(s) within the plant provided that the overall expression in theplant only affects (or significantly affects) outgrowth from meristems.

The promoter according to the present invention may in addition to beingoperable to direct expression in the lateral bud and/or lateral shootmay also be operable to direct expression in other cell type(s) ortissue(s) within the plant provided that the overall expression does notkill the plant.

Preferably, the term “substantially specifically” as used herein meansthat the promoter according to the present invention is operable todirect expression predominantly in lateral buds and/or lateral shoots ofa plant such that, for instance, a nucleic acid sequence operablyassociated with said promoter is expressed predominantly in lateral budsand/or lateral shoots with less than 50%, preferably less than 25%,preferably less than 10%, more preferably less than 5% of the overallexpression level of said nucleic acid sequence being in any other planttissue or cell of the plant.

For example, a nucleic acid sequence expressed under the control of thepromoter according to the present invention may be predominantlyexpressed in the lateral bud and/or lateral shoot with less than 25%,preferably less than 10%, more preferably less than 5% of the overallexpression level in any other tissue.

The promoter sequence may comprise the sequence shown in SEQ ID No.1,SEQ ID No. 7 or SEQ ID No.4, or a functional part thereof, or a sequencehaving at least 60% identity thereto.

Preferably the promoter sequence is the sequence shown in SEQ ID No.1,SEQ ID No. 7 or SEQ ID No.4, or a functional part thereof, or a sequencehaving at least 60% identity thereto.

Preferably, the promoter sequence comprises the sequence shown in SEQ IDNo.1 or a functional part thereof or a sequence which is at least 75%,preferably at least 80%, preferably at least 90%, preferably at least97%, identical thereto.

Preferably, the promoter sequence comprises the sequence shown in SEQ IDNo. 7 or a functional part thereof or a sequence which is at least 75%,preferably at least 80%, preferably at least 90%, preferably at least97% identical thereto.

In one embodiment of the invention the morphology of a plant is affectedby one or more gene products, preferably one gene product.

In a further embodiment of the invention the morphology of a plant isaffected by two or more gene products. Preferably the nucleic acidsequence encodes two or more gene products. Thus, by way of example onlyone chimaeric gene may be introduced into the plant, which chimaericgene comprises a nucleic acid sequence encoding two or more geneproducts. Alternatively two or more chimaeric genes may be introducedinto the plant, each chimaeric gene comprising a nucleic acid sequenceencoding a gene product. By way of example only, two or more chimaericgenes may be introduced into the plant, each chimaeric gene comprising anucleic acid sequence encoding one or more gene products. Thus, forexample, one chimaeric gene may comprise a nucleic acid sequenceencoding one gene product and a further chimaeric gene may comprise anucleic acid sequence encoding multiple gene products, i.e. 2, 3 or 4gene products. Alternatively, both chimaeric genes may comprise nucleicacid sequence(s) encoding multiple gene products. Preferably the two ormore gene products function independently of each other to effectdisruption of the metabolism of the cells by an additive effect.Alternatively the two or more gene products interact with each other toeffect disruption of the metabolism of the cells by a synergistic or byan antagonistic effect.

Preferably the outgrowth of lateral shoots is modified. The outgrowth oflateral shoots may be enhanced. Suitably the outgrowth of lateral shootsmay be prevented or reduced and/or delayed.

Preferably the outgrowth of lateral shoots is modified by disruptingmetabolism or by causing death of cells involved in lateral buddevelopment.

Cells involved in the lateral bud development may include those of thepro-meristem, protoderm, epidermis, stomata, guard cells, endodermis,periderm, cortex parenchyma, stinging cells, storage cells, ovuleendosperm, pollen, abscission zone, trichomes, secretory cells, phellem,phellogen, phelloderm, procambium, cambium, protoxylem, xylem, rays,protophloem, phloem, collenchyma, sclerenchyma, parenchyma,chlorenchyma, tunica, corpus, cortex, prophyll structures, and foliarstructures.

The term “specific cells” as used herein is intended to mean those cellsin which the promoter is predominantly expressed, preferably those cellsin which the promoter is expressed substantially specifically.

The term “specific cells of the plant” includes lateral bud and/orlateral shoot cells, i.e. those cells involved in lateral bud initiationand/or development. In one embodiment preferably the “specific cells ofthe plant” are the lateral bud and/or lateral shoot cells.

In one embodiment the specific cells of the plant may be a cell of oneor more of the: pro-meristem, protoderm, epidermis, stomata, guardcells, endodermis, periderm, cortex parenchyma, stinging cells, storagecells, ovule endosperm, pollen, abscission zone, trichomes, secretorycells, phellem, phellogen, phelloderm, procambium, cambium, protoxylem,xylem, rays, protophloem, phloem, collenchyma, sclerenchyma, parenchyma,chlorenchyma, tunica, corpus, cortex, prophyll structures, and foliarstructures.

The term “predominantly expressed” as used herein means that thepromoter according to the present invention is operable to mainly(i.e >51% of the total expression) direct expression in the specificcells of the plant (such as the later bud and/or lateral shoot cells),although lower levels of expression may be found in other cell type(s)or tissue(s).

For example, it has been found that the promoter according to thepresent invention is operable to predominantly direct expression in thelateral bud and/or lateral shoot but that there may also be expressionin another tissue or cell type. For example, there may also beexpression in wound tissue and/or stem and/or leaf tissue.

Suitably the outgrowth of the lateral bud or lateral shoot may beprevented or reduced and/or delayed. Preferably the outgrowth of thelateral bud or lateral shoot is modified by disrupting metabolism or bycausing death of cells involved in lateral bud and/or lateral shootdevelopment.

The term “lateral bud tissue” as used herein includes lateral bud cellsand/or lateral shoot cells.

Preferably the gene product of the nucleic acid sequence capable ofdisrupting the metabolism or of causing death of specific cells and/ornearby cells is a deleterious product, suitably a cytotoxic molecule.

Preferably the gene product of the nucleic acid sequence capable ofdisrupting the metabolism or of causing death of specific cells and/ornearby cells is a ribosome inactivating protein (RIP) or a variant orfunctional part thereof. For example, the RIP may be a Type 1 RIP and/ora Type 2 RIP and/or a Type 3 RIP or a variant or functional partthereof. Preferably the gene product is a pokeweed antiviral protein(PAP) or a variant or functional part thereof. The gene product may bePAP′ or PAPII or a variant or functional part thereof. More preferablythe gene product is pokeweed antiviral protein S (PAP-S) or a variant orfunctional part thereof.

The present invention also provides a nucleic acid comprising a promotersequence, the promoter sequence being as shown in SEQ ID No. 1, orfunctional part thereof, or a sequence having at least 65% identitythereto, more preferably at least 75% identity thereto, more preferablyat least 85% identity thereto, more preferably at least 95% identitythereto, more preferably at least 97% identity thereto, more preferablyat least 98% identity thereto, most preferably at least 99% identitythereto and being capable of regulating expression of a gene.

The present invention also provides a nucleic acid comprising a promotersequence, the promoter sequence being as shown in SEQ ID No. 7, orfunctional part thereof, or a sequence having at least 65% identitythereto, more preferably at least 75% identity thereto, more preferablyat least 85% identity thereto, more preferably at least 95% identitythereto, more preferably at least 97% identity thereto, more preferablyat least 98% identity thereto, most preferably at least 99% identitythereto and being capable of regulating expression of a gene.

The present invention also provides a chimaeric gene comprising apromoter sequence operably associated with a nucleic acid sequence thepromoter sequence being as shown in SEQ ID No. 1, or functional partthereof, or a sequence having at least 65% identity thereto morepreferably at least 75% identity thereto, more preferably at least 85%identity thereto, more preferably at least 95% identity thereto, morepreferably at least 97% identity thereto, more preferably at least 98%identity thereto, most preferably at least 99% identity thereto andbeing capable of regulating expression of a gene.

The present invention also provides a chimaeric gene comprising apromoter sequence operably associated with a nucleic acid sequence thepromoter sequence being as shown in SEQ ID No. 7, or functional partthereof, or a sequence having at least 65% identity thereto, morepreferably at least 75% identity thereto, more preferably at least 85%identity thereto, more preferably at least 95% identity thereto, morepreferably at least 97% identity thereto, more preferably at least 98%identity thereto, most preferably at least 99% identity thereto andbeing capable of regulating expression of a gene.

The present invention also provides a chimaeric gene comprising apromoter sequence operably associated with a nucleic acid sequence thepromoter sequence being as shown in SEQ ID No. 4, or a functional partthereof, or a sequence having at least 65% identity thereto and beingcapable of regulating expression of a gene.

Preferably the nucleic acid sequence and/or the nucleic acid accordingto the present invention and/or the chimaeric gene according to thepresent invention is a DNA sequence.

In one embodiment the chimaeric gene according to the present inventionis obtainable, preferably obtained, from the clone pBNP 085-0501-001(NCIMB 41343).

Preferably the nucleic acid sequence is capable of regulating expressionof an additional sequence. Suitably the additional sequence encodes aprotein or RNA, a cosuppression sequence, an antisense sequence or adsRNA (double-stranded RNA) inhibition sequence.

Preferably the additional sequence is obtainable, preferably obtained,from a plant. The plant may be a member of the family Solanaceae.Preferably the plant may be a member of the subfamily Cestroideae. Morepreferably the plant is one or more of tomato, potato, aubergine,Petunia or tobacco. More preferably the plant is from the genusNicotiana. Most preferably the plant is Nicotiana tabacum.

It is preferred that the additional sequence is capable of disruptingthe metabolism of or causing death of the specific cells and/or nearbycells. It is preferred that the additional sequence encodes a pokeweedantiviral protein or functional part thereof.

The nucleic acid sequences and/or nucleic acid and/or the chimaeric genereferred to herein may be isolated sequences or, alternatively, may besynthesised sequences.

The present invention further provides a recombinant DNA comprisingvector DNA and a nucleic acid sequence according to the presentinvention and/or a nucleic acid according to the present inventionand/or a promoter sequence according to the present invention and/or achimaeric gene according to the present invention. The recombinant DNAmay suitably further comprise a coding sequence of a gene. Preferablythe vector DNA comprises a plasmid, cosmid, virus or phage. Suitably therecombinant DNA may comprise a promoter to direct expression of aselectable marker gene.

Preferably the recombinant DNA resides in a host cell. Suitably the hostcell may allow transcription and translation of the recombinant DNA.

The present invention further provides a plant produced according to themethod of the present invention. The present invention further providesa genetically-engineered plant comprising a nucleic acid sequence and/ora nucleic acid and/or a promoter sequence and/or a chimaeric geneaccording to the invention. Preferably the nucleic acid and/or apromoter sequence is operably associated with a coding sequence of agene.

According to a further aspect of the present invention there is provideda plant comprising a recombinant DNA according to the present invention.The plant according to the present invention may be of interest to thehorticulture industry, the floriculture industry, the forestry industryand/or the agriculture industry. The plant may be a plant which is grownfor the purpose of providing cut flowers. The plant may be tomato,cucumber, Petunia, Dianthus, Picea, Pinus, Eucalyptus, Populus, adicotyledonous species such as potato, tobacco, cotton, lettuce,eggplant, melon, squash, pea, canola, soybean, sugar beet or sunflower,or a monocotyledonous species such as wheat, barley, rye, rice, ormaize. More preferably the plant is of the family Solanaceae. Morepreferably the plant is of the subfamily Cestroideae. More preferablythe plant is one or more of tomato, potato, aubergine, Petunia ortobacco. More preferably the plant is of the genus Nicotiana. Mostpreferably the plant is Nicotiana tabacum.

The present invention even further provides a plant cell from a plantproduced according to the method of the present invention, the plantcell having an altered metabolism. The plant cell produced according tothe method of the present invention may have a metabolism which isaltered to enhance its metabolism. Preferably, the plant cell producedaccording to the present invention has a disrupted metabolism.

The present invention also provides a genetically-engineered plant cellcomprising a nucleic acid sequence according to the present invention.Preferably the nucleic acid according to the present invention and/orthe promoter sequence according to the present invention is operablyassociated with a coding sequence of a gene.

In an even further aspect of the present invention there is provided aplant cell comprising a recombinant DNA according to the presentinvention. Preferably the nucleic acid according to the presentinvention and/or the promoter sequence according to the presentinvention is operably associated with a coding sequence of a gene.

The present invention also provides a method of regulating theexpression of a gene in a plant, the method comprising introducing intothe plant a nucleic acid according to the present invention and/or apromoter sequence according to the present invention operably associatedwith a coding sequence of a gene the expression of which gene is to beregulated.

The present invention further provides a method of modifying themetabolism within a cell of a transgenic plant, the method comprisingintroducing into a plant a nucleic acid according to the presentinvention and/or a promoter sequence according to the present inventionand/or a chimaeric gene according to the present invention. Preferablythe method comprises introducing a nucleic acid according to the presentinvention and/or a promoter sequence according to the present inventionand/or a chimaeric gene according to the present invention into thecell. Preferably the nucleic acid or the promoter sequence according tothe present invention is operably associated with a coding sequence of agene. Advantageously the gene is involved in a metabolic pathway.Preferably a metabolic product is increased or decreased in the cell.

The present invention even further provides a method of alteringproduction of a gene product within a plant cell comprising introducinga nucleic acid according to the present invention and/or a promotersequence according to the present invention operably associated with acoding sequence of a gene, the production of the gene product of whichgene is to be altered. Preferably production of the gene product isincreased. Preferably the gene product is an RNA molecule which caninteract with the gene expression process via the mechanism of RNAi,antisense, or cosuppression, or which can be translated into a proteingene product. Preferably the protein gene product is a protease,restriction endonuclease, membrane transport protein, ribonuclease orribosome-inactivating protein. Preferably the ribosome-inactivatingprotein is pokeweed antiviral protein (PAP). The PAP may be PAP′ orPAPII. More preferably the pokeweed antiviral protein is PAP-S or avariant or functional part thereof.

Further provided by the present invention is the use of a nucleic acidcomprising a promoter sequence as shown in SEQ ID No.1 or SEQ ID No. 7,or functional part thereof, or a sequence having at least 60% identitythereto for regulating expression of a gene in a plant.

Also provided by the present invention is the use of a nucleic acidsequence comprising the promoter sequence shown in SEQ ID No. 1 orfunctional part thereof or a sequence having at least 75%, preferably atleast 80%, preferably at least 90%, preferably at least 97%, identitythereto, for regulating the expression of a gene in a plant.

Also provided by the present invention is the use of a nucleic acidsequence comprising the promoter sequence shown in SEQ ID No. 7 orfunctional part thereof or a sequence having at least 75%, preferably atleast 80%, preferably at least 90%, preferably at least 97%, identitythereto, for regulating the expression of a gene in a plant.

Also provided by the present invention is the use of a nucleic acidcomprising a promoter sequence as shown in SEQ ID No.1 or SEQ ID No.7 orSEQ ID No.4, or functional part thereof, or a sequence having at least60% identity thereto for altering metabolism within a plant cell.

Also provided by the present invention is the use of a nucleic acidsequence comprising a promoter sequence as shown in SEQ ID No. 1 orfunctional part thereof or a sequence which is at least 75%, preferablyat least 80%, preferably at least 90%, preferably at least 97%,identical thereto for altering metabolism within a plant cell.

Also provided by the present invention is the use of a nucleic acidsequence comprising a promoter sequence as shown in SEQ ID No. 7 orfunctional part thereof or a sequence which is at least 75%, preferablyat least 80%, preferably at least 90%, preferably at least 97%,identical thereto for altering metabolism within a plant cell.

The present invention even further provides the use of a nucleic acidcomprising a promoter sequence as shown in SEQ ID No.1 or SEQ ID No. 7or SEQ ID No.4, or functional part thereof, or a sequence having atleast 60% identity thereto for altering production of a gene productwithin a plant cell.

Also provided by the present invention is the use of a nucleic acidsequence comprising a promoter sequence shown as SEQ ID No. 1 orfunctional part thereof or a sequence which is at least 75%, preferablyat least 80%, preferably at least 90%, preferably at least 97%,identical thereto for altering production of a gene product within aplant cell.

Also provided by the present invention is the use of a nucleic acidsequence comprising a promoter sequence shown as SEQ ID No. 7 orfunctional part thereof or a sequence which is at least 75%, preferablyat least 80%, preferably at least 90%, preferably at least 97%,identical thereto for altering production of a gene product within aplant cell.

The present invention also provides an oligonucleotide probe whichselectively hybridizes to a nucleic acid sequence according to thepresent invention.

The present invention may also provide a part of SEQ ID No. 1, whereinthe part is from nucleotide 1 to nucleotide 1321 of SEQ ID No.1 or apart thereof. The part from nucleotide 1 to nucleotide 1321 may be a“functional part” of SEQ ID No. 1.

The present invention may also provide a part of SEQ ID No. 7, whereinthe part is from nucleotide 1 to nucleotide 1309 of SEQ ID No. 7 or apart thereof. The part from nucleotide 1 to nucleotide 1309 may be a“functional part” of SEQ ID No. 7.

By “functional part” we mean the part is operable to direct expressionin specific cells of a plant.

Any aspect of the present invention may relate to only one, or more thanone, of the sequences referred to herein.

The term “chimaeric gene” as used herein means any hybrid nucleic acidmolecule formed when nucleic acid sequences from different sources areligated together. In the present invention “nearby cells” are thosecells that are sufficiently close to the specific cells that they areaffected by expression in the specific cells, i.e. “nearby cells” may bedefined as cells which may differentiate into lateral bud tissue (i.e.once lateral bud initiation cells have been altered in accordance withthe present invention), or cells which support the growth of the lateralbud and/or lateral shoot.

SEQUENCE IDENTIFIERS

In the sequence listing:

SEQ ID No. 1 shows the DNA sequence of an isolated promoter of thepresent invention.

SEQ ID No. 2 shows the PCR Oligonucleotide S2PCLOFWD.

SEQ ID No. 3 shows the PCR Oligonucleotide S2PCLOREV.

SEQ ID No.4 shows the DNA sequence of a further isolated promoter of thepresent invention (known herein as the “ATC 023 promoter”).

SEQ ID No. 5 shows the PCR Oligonucleotide AT4G29190L.

SEQ ID No. 6 shows the PCR Oligonucleotide AT4G29190R.

SEQ ID No. 7 show the DNA sequence of an isolated promoter of thepresent invention (known herein as the “ATC 085 promoter”).

SEQ ID No. 8 shows the Sar8.2b promoter (Genbank accession U64816).

SEQ ID No. 9 shows a region of Arabidopsis thaliana genome around thelocus AT4g29190 [Chromosome 4: 14396379-14392944 reverse orientation].

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be readily carried into effect referencewill now be made, by way of example, to the following drawings in which;

FIG. 1A through 1E show the alignment of SEQ ID No. 1 with SEQ ID No.8,the Sar8.2b promoter (Genbank accession U648116).

FIG. 2A through 2G show the alignment of SEQ ID No. 4, the ATC 023promoter sequence, with SEQ ID No. 9, the Arabidopsis thaliana genome,around the locus AT4g29190 [Chromosome 4: 14396379-14392944 reverseorientation]. The 5′ upstream region of the AT4g29190 gene is shown initalics, and the 5′ and 3′ untranslated regions are shown in lowercaselettering. Boxes indicate the initiation and termination codons of theAT4g29190 gene. The primer sites used for cloning the ATC 023 promoterare underlined. Nucleotide changes are shown double underlined.

FIG. 3 shows the location of expression of the GUS reporter gene drivenby the ATC 085 promoter in tobacco stem sections. (a)-(c) show sectionsthrough stem at the region of lateral bud initiation; (d) shows threeserial sections through the same lateral bud initiation region; and (e)shows a vertical section through a leaf axil.

FIG. 4a shows the effect of expression of the Pokeweed antiviral proteindriven by the ATC 085 promoter on the outgrowth of lateral buds 17 and18 in tobacco following topping compared with control (NCC) plants.

FIG. 4b shows the effect of expression of the Pokeweed antiviral proteindriven by the ATC 085 promoter on the outgrowth of lateral bud 16 intobacco following topping compared with control (NCC) plants.

FIG. 5 shows the outgrowth of lateral shoots in tobacco upon topping,and the effect of transformation with ATC 085-PAP; (a) shows anuntransformed control plant, showing elongated outgrowth of lateralshoots; (b) shows an ATC085-PAP transgenic plant showing no visible budoutgrowth.

FIG. 6 shows the location of expression of the GUS reporter gene drivenby the ATC 023 promoter in tobacco stem sections following topping. (a)shows a section through stem of one plant at the region of lateral budinitiation; (b) and (c) show serial sections through two lateral budinitiation regions taken from different plants; and (d) shows a verticalsection through a leaf axil.

FIG. 7 shows the location of expression of the GUS reporter gene drivenby the ATC 023 promoter in Arabidopsis stem sections. (a) showsexpression at the point of bud outgrowth early in bud development, and(b)-(d) show subsequent expression in developing buds and nearbytissues.

DETAILED DESCRIPTION OF THE INVENTION

Suitably, the promoter sequence according to the present inventioncomprises a nucleotide sequence which has at least 65%, preferably atleast 70%, more preferably at least 75%, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 95%, more preferably at least 97% identity, morepreferably at least 98% identity, most preferably at least 99% identitywith any one of the sequences shown as SEQ ID No. 1 or SEQ ID No. 7 or afunctional part thereof.

The term “promoter” as used herein is used in the normal sense of theart, e.g. an RNA polymerase binding site.

As used herein, “altering the metabolism” of a cell means affecting themetabolic function of a cell in such a way as to change the normalfunctioning metabolism of the cell, resulting in either enhanced orinhibited normal function of the cell.

As used herein, the term “disrupting the metabolism” means altering themetabolic function of a cell in such a way as to interfere with thenormal functioning metabolism of the cell, resulting in the death orinhibition of the normal function of the cell.

As used herein, the term “enhancing the metabolism” of a cell meansaltering the metabolic function of a cell in such a way as to increasethe growth or viability of the cell.

As used herein, “lateral bud initiation cell” means a cell associatedwith the initiation of the growth of a lateral bud.

As used herein, “modifying morphology” means altering the normal growthhabit of a plant which manifests as a physical change to part or all ofthe plant. The growth of the lateral shoots of the plant may beenhanced, for example. Preferably the growth of the lateral shoots ofthe plant is inhibited or prevented. Either way the overall physicalstructure and/or appearance of the plant may be changed.

A plant DNA sequence may be recovered from the cells of the naturalhost, or it may be synthesized directly in vitro. Extraction from thenatural host enables the isolation de novo of novel sequences, whereasin vitro DNA synthesis generally requires pre-existing sequenceinformation. Direct chemical in vitro synthesis can be achieved bysequential manual synthesis or by automated procedures. DNA sequencesmay also be constructed by standard techniques of annealing and ligatingfragments, or by other methods known in the art. Examples of suchcloning procedures are given in Sambrook et al. (1989).

The DNA sequence of the present invention may be isolated by directcloning of segments of plant genomic DNA. Suitable segments of genomicDNA may be obtained by fragmentation using restriction endonucleases,sonication, physical shearing, or other methods known in the art. Byusing predictive screening of the DNA sequence of the cloned segment forthe presence of coding sequences (Baxevanis, 2001) motifs characteristicof known promoter sequences may be found upstream of such a diagnosticsequence.

The identification of the cloned segment as a promoter sequence mayalternatively be achieved by assessing functionality, for example bylinking the cloned segment with a coding sequence derived from areporter gene and introducing the chimaeric construct into a host cellor cell-free system wherein expression of the reporter gene can beevaluated. This process may form part of another sequence isolationstrategy termed promoter trapping, wherein genomic DNA fragments arecloned directly into “expression vectors” comprising a reporter genecoding region and other sequences necessary for expression in a hostcell or cell-free system. The expression may or may not requireintegration of the chimaeric construct into the host's chromosomal DNA.

An alternative method of obtaining a DNA sequence of the presentinvention is by the identification and isolation of a DNA codingsequence which is known to be expressed and subsequently using thissequence to obtain the contiguous promoter sequence, which is bydefinition directing the expression of the coding sequence.Alternatively a DNA sequence may be obtained by identification of asequence which is known to be expressed in a different organism, andthen isolating the homologous coding sequence and subsequently itsassociated promoter sequence from the organism of choice. A codingsequence may be obtained by the isolation of messenger RNA (mRNA orpolyA+RNA) from plant tissue or isolation of a protein and performing“back-translation” of its sequence. The tissue used for RNA isolation isselected on the basis that suitable gene coding sequences are believedto be expressed in that tissue at optimal levels for isolation.

Various methods for isolating mRNA from plant tissue are well known tothose skilled in the art, including for example using an oligo-dToligonucleotide immobilised on an inert matrix. The isolated mRNA may beused to produce its complementary DNA sequence (cDNA) by use of theenzyme reverse transcriptase (RT) or other enzymes having reversetranscriptase activity. Isolation of an individual cDNA sequence from apool of cDNAs may be achieved by cloning into bacterial or viralvectors, or by employing the polymerase chain reaction (PCR) withselected oligonucleotide primers. The production and isolation of aspecific cDNA from mRNA may be achieved by a combination of the reversetranscription and PCR steps in a process known as RT-PCR.

Various methods may be employed to improve the efficiency of isolationof the desired sequence through enrichment or selection methodsincluding the isolation and comparison of mRNA (or the resulting singleor double-stranded cDNA) from more than one source in order to identifythose sequences expressed predominantly in the tissue of choice.Numerous methods of differential screening, hybridisation, or cloningare known to those skilled in the art including cDNA-AFLP, cascadehybridisation, and commercial kits for selective or differentialcloning.

In the present invention, a cDNA sequence to a SAR 8.2j protein (EMBLAccession Number U64812) was utilised to isolate a novel promotersequence. Examples of other SAR protein genes are given in EMBLAccession Nos. U64816, U64807, U64808, U64809, U64810, U64811, U64813,U64814, U6481. A second cDNA sequence to a Zinc Finger TranscriptionFactor (EMBL Accession Number AL096692) was also utilised to isolate apromoter sequence.

The selected cDNA may then be used to evaluate the genomic features ofits gene of origin, by use as a hybridisation probe in a Southern blotof plant genomic DNA to reveal the complexity of the genome with respectto that sequence. Alternatively, sequence information from the cDNA maybe used to devise oligonucleotides and these can be used in the same wayas hybridisation probes; for PCR primers to produce hybridisationprobes, or for PCR primers to be used in direct genome analysis.

Similarly the selected cDNA may be used to evaluate the expressionprofile of its gene of origin, by use as a hybridisation probe in aNorthern blot of RNA extracted from various plant tissues, or from adevelopmental or temporal series. Again sequence information from thecDNA may be used to devise oligonucleotides which can be used ashybridisation probes, to produce hybridisation probes, or directly forRT-PCR.

The selected cDNA, or derived oligonucleotides, may then be used as ahybridisation probe to challenge a library of cloned genomic DNAfragments and identify overlapping DNA sequences. By this means acontiguous promoter may be identified and isolated.

By the nature of the method of isolation, an isolated cDNA usuallycomprises the 3′ terminus of the coding region and extends towards the5′ terminus. It may not comprise the full-length coding sequence. It ispreferable to ensure that the 5′ terminal sequence is present if thecDNA is to be used to identify the contiguous promoter. This may beachieved by extension of the cloned cDNA sequence in the 5′ direction bya process termed 5′ RACE (rapid amplification of cDNA ends).

If sequence analysis of the cloned cDNA identifies a homologous sequencealready reported in the scientific literature, this information mayprovide a suitable candidate sequence for the 5′ terminus. However, thepossibility of there being different members of the same gene familywith similar coding regions, but differing intron regions, promotersequences and expression profiles, may lead to the selection of anincorrect and unsuitable promoter sequence.

Once the 5′ terminus of the coding sequence has been identified, thecontiguous upstream region containing the promoter may be identified ifpresent in the public Nucleotide databases. Alternatively the promotermay be isolated, by further extension in the 5′ direction. This may beachieved by methods including vector-ligation PCR, genome walking,vectorette PCR, and other methods. If necessary the process may berepeated with a new primer complementary to the 5′ terminus of the firstpromoter fragment to ensure that all the control sequences of thepromoters are isolated.

The present invention also provides for the use of a DNA sequence asshown in SEQ. ID. No: 4, the sequence encoding a promoter (and referredto herein as “ATC 023”), the sequence being able to control theexpression of a Zinc finger transcription factor protein, and which maybe used as a plant promoter. This sequence is identified on the TAIRNucleotide Sequence database at Locus AT4g29190. The present inventionalso includes DNA sequences which control the expression of other Zincfinger transcription factor protein genes and which have some regions ofhomology with the DNA sequence of SEQ. ID. No: 4.

Homology may be determined on the basis of percentage identity betweentwo DNA (or polypeptide) sequences. In general the two sequences to becompared are aligned to give a maximum correlation between thesequences. The alignment of the two sequences is examined and the numberof positions giving an exact nucleotide (or amino acid) correspondencebetween the two sequences determined, divided by the total length of thealignment multiplied by 100 to give a percentage identity figure. Thispercentage identity figure may be determined over the whole length ofthe sequences to be compared, which is particularly suitable forsequences of the same or very similar lengths and which are highlyhomologous, or over shorter defined lengths, which is more suitable forsequences of unequal length or which have a lower level of homology.

Methods for comparing the identity of two or more sequences are wellknown in the art. Thus for instance, programs available in the WisconsinSequence Analysis Package, version 9.1 (Devereux J. et al, 1984)(available from Genetics Computer Group, Madison, Wis., USA), forexample the programs BESTFIT and GAP, may be used to determine thepercentage identity between two polynucleotides and the percentageidentity between two polypeptide sequences. BESTFIT uses the “localhomology” algorithm of Smith and Waterman (1981) and finds the bestsingle region of similarity between two sequences. BESTFIT is moresuited to comparing two polynucleotide or two polypeptide sequenceswhich are dissimilar in length, the program assuming that the shortersequence represents a portion of the longer. In comparison, GAP alignstwo sequences finding a “maximum similarity” according to the algorithmof Needleman and Wunsch (1970). GAP is more suited to comparingsequences which are approximately the same length and an alignment isexpected over the entire length. Preferably the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3 for polynucleotidesequences and 12 and 4 for polypeptide sequences, respectively.Preferably, percentage identities and similarities are determined whenthe two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Karlin & Altschul, 1990, Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in Karlin & Altschul, 1993, Proc. Natl. Acad.Sci. USA 90:5873-5877, available from the National Center forBiotechnology Information (NCBI), Bethesda, Md., USA and accessiblethrough the home page of the NCBI at www.ncbi.nlm.nih.gov). Theseprograms exemplify a preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al., 1997, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program to obtain nucleotidesequences homologous to a nucleic acid molecule of the invention. BLASTprotein searches can be performed with the XBLAST program, to obtainamino acid sequences homologous to a protein molecule of the invention.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-Blast can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov. Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage.

Another non-limiting example of a program for determining identityand/or similarity between sequences known in the art is FASTA (PearsonW. R. and Lipman D. J., Proc. Nat. Acac. Sci., USA, 85:2444-2448, 1988,available as part of the Wisconsin Sequence Analysis Package).Preferably the BLOSUM62 amino acid substitution matrix (Henikoff S. andHenikoff J. G., Proc. Nat. Acad. Sci., USA, 89:10915-10919, 1992) isused in polypeptide sequence comparisons including where nucleotidesequences are first translated into amino acid sequences beforecomparison.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

Preferably the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to apolynucleotide or a polypeptide sequence of the present invention, thequery and the reference sequence being optimally aligned and theparameters of the program set at the default value.

In the context of the present invention substantially homologoussequences are those which have at least 50% sequence identity,preferably at least 60%, 65% or 70% sequence identity, more preferablyat least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% sequenceidentity and most preferably at least 95%, 96%, 97%, 98% or 99% or abovesequence identity.

As used herein, the term “homologue” means an entity having a certainhomology with the subject amino acid sequences and the subjectnucleotide sequences. Here, the term “homology” can be equated with“identity”.

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present invention it is preferred to express homologyin terms of sequence identity.

In the context of the present invention the term utilising includes,amongst others, using, introducing and expressing a sequence or gene.

The present invention also includes DNA sequences which hybridize to theDNA sequences of the above promoters, including partial sequences andcomplementary sequences. Conditions under which such sequences will sohybridize can be determined in a routine manner.

Hybridization can be performed under low, medium or high stringencyconditions. The conditions under which hybridization and/or washing canbe carried out can range from 42° C. to 68° C. and the washing buffercan comprise from 0.1×SSC, 0.5% SDS to 6×SSC, 0.5% SDS. Typically,hybridization can be carried out overnight at 65° C. (high stringencyconditions), 60° C. (medium stringency conditions), or 55° C. (lowstringency conditions). The filters can be washed for 2×15 minutes with0.1×SSC, 0.5% SDS at 65° C. (high stringency washing). The filters maybe washed for 2×15 minutes with 0.1×SSC, 0.5% SDS at 63° C. (mediumstringency washing). For low stringency washing, the filters are washedat 60° C. for 2×15 minutes at 2×SSC, 0.5% SDS.

The present invention also includes DNA sequences which hybridize tooligonucleotide probes. Preferably the DNA sequences hybridize tooligonucleotide probes under stringent conditions. In instances whereinthe nucleic acid molecules are oligonucleotides (“oligos”), highlystringent conditions may refer, for example, to washing in 6×SSC/0.05%sodium pyrophosphate at 37° C. (for 14 base oligos), 48° C. (for 17 baseoligos), 55° C. (for 20 base oligos), and 60° C. (for 23 base oligos).In a BLAST search against the EMBL Nucleotide Sequence Database, release77 (Kulikova et al, 2004), the ATC 085 promoter has a greater than 90%identity over its last 220 bases with known sequences upstream of thetranslation start codon of other SAR genes such as 8.2h and 8.2k. Theonly sequence showing any other regions of homology along the length ofthe promoter is the Sar 8.2b promoter region (SAR 8.2b promoter, Genbankaccession U64816, bases 1 to 1907).

FIG. 1A through 1E show the alignment between SEQ ID No. 1 (bases 1 to1566) and the region of the SAR 8.2b promoter with homology (bases 704to 1907 of the Genbank sequence). Identity is shown by dots between thealigned sequences. From FIG. 1A through 1E, it can be seen that SEQ IDNo. 1 has a 61% identity with the corresponding SAR 8.2b promoteroverall.

FIG. 2A through 2G show the alignment between the sequence of the ATC023 promoter (bases 1 to 1904) and the region of the Arabidopsisthaliana sequence around the locus AT4g29190 (in reverse orientation).From FIG. 2A through 2G, it can be seen that the ATC 023 promoter ascloned has over 99% identity with the reported genomic sequence.

The gene coding sequence that is used under the control of the promoterand employed in carrying out the present invention may be active in someor all plant tissues. The sequence employed may encode a protein or anRNA moiety. Through recombinant DNA techniques the sequence may encode asynthetic variant of a protein or RNA, a partial sequence, or acomposite sequence comprising regions from one or more genes. Forexample, the chimaeric gene may encode a polyprotein. The sequence mayalso comprise repeated, truncated, inverted or complementary sequences,to achieve disruption of the transcription and translation of anendogenous gene or genes by, for example, antisense, cosuppression, orRNA inhibition technology.

Many plant, bacterial and viral genes may be actively expressed underthe control of the promoter. Preferably such genes encode:

-   -   (i) GUS, GFP and luciferase enzymes which may be used as        reporter genes for promoter function.    -   (ii) Genes for enzymes producing structural components of the        plant such as cellulose, hemicelluloses, pectins and lignin.    -   (iii) DNA sequences designed to alter the metabolism in plants        and plant cells. Such genes include those for carbohydrate        metabolism, starch metabolism, amino acid and protein        metabolism, nucleic acid metabolism and lipid metabolism.    -   (iv) Regulatory DNA sequences which may have effects on the        control of metabolism or development, such as flowering or plant        architecture, including those genes involved in the metabolism        or transport of plant growth substances.    -   (v) DNA sequences encoding products such as restriction enzymes,        proteases, proteinase inhibitors and ribosome inactivating        proteins which can be used to deliberately impair cell function.    -   (vi) Resistance to environmental stress, or to pathogens or to        pests.

The promoter of the present invention is preferably operable to directexpression in lateral and/or auxiliary meristems.

The promoter according to the present invention may in addition to beingoperable to direct expression in lateral buds and/or shoots, be operableto direct expression in other tissues and/or cells of the plant. Forinstance, some expression may be observed in wound tissue, and/or thestem and/or the leaves of the plant. However, the promoter is operableto direct expression substantially specifically (as defined herein) in alateral bud and/or lateral shoot.

The expression profile of the promoters of the present invention may bedetermined by those skilled in the art using a variety of methods whichare preferably:

-   -   (i) Transient expression where the promoter is linked to a        reporter gene such as GUS, GFP or luciferase in an appropriate        construct and introduced into plant tissue by shotgun        transformation. Promoter expression is detected by the presence        or absence of the reporter gene product in different plant        tissues.    -   (ii) Transient expression where the promoter is linked to a        reporter gene such as GUS, GFP or luciferase in an appropriate        binary construct and introduced into plant tissue by        Agrobacterium. Promoter expression is detected by the presence        or absence of the reporter gene product in different plant        tissues. The GUS activity can be detected visually using        appropriate substrates as a stain, or by using a colorimeter or        spectrophotometer; luciferase and fluorescent marker proteins        can be detected by eye or photographically, either on film or        digital medium, or quantitatively using a luminometer or        fluorometer. Marker genes may also be detected using antibody        based systems.    -   (iii) Plant transformation and regeneration. Promoter expression        is detected by the presence or absence of the reporter gene in        different plant tissues.    -   (iv) Plant transformation and regeneration. Promoter expression        is detected by the presence or absence of a specific gene        product produced by an effector gene in different plant tissues.

The gene product of the present invention may be produced by recombinanttechniques, wherein genomic DNA clones or cDNA clones for the DNA codingsequence are produced, isolated, proliferated, and incorporated into aplant transformation vector of the present invention.

The present application is the first time that a promoter which iscapable of expressing a gene substantially specifically in lateral budand/or lateral shoots has been presented.

Suitably, the promoter according to the present invention is an “inducedpromoter”, i.e. one which is switched on in response to a stimulus.Thus, as well as the promoter being operable to direct expression inspecific cells of the plant, the promoter may also be considered as aninducible promoter.

Preferably, the promoter according to the present invention is inducedby topping the plant, i.e. removing the apical bud or shoot of theplant.

Without wishing to be bound by theory, one possible manner in which thepromoter of the present invention may be “switched on” is that it may beactivated or induced by a signal which may be stimulated by the removalof the apical bud, thus after topping. For instance, the signal may be aplant growth hormone. By way of example only the signal may be acytokinin(s).

Alternatively, again without being bound by theory, it may be possiblethat the apical bud releases an inhibitory signal which prevents lateralmeristem outgrowth and/or the switching on the promoter of the presentinvention. In which case, for example, the removal of the apical bud,e.g by topping, may result in the loss of the inhibitory signal and thusthe switching on of the promoter of the present invention. For instance,by way of examply only, the inhibitory signal may be a auxin(s).

Isolated

In one aspect, preferably the sequence is in an isolated form. The term“isolated” means that the sequence is at least substantially free fromat least one other component with which the sequence is naturallyassociated in nature and as found in nature.

Purified

In one aspect, preferably the sequence is in a purified form. The term“purified” means that the sequence is in a relatively pure state—e.g. atleast about 90% pure, or at least about 95% pure or at least about 98%pure.

Nucleotide Sequence

The term “nucleotide sequence” as used herein is synonymous with“nucleic acid sequence” and refers to an oligonucleotide sequence orpolynucleotide sequence, and variants, homologues, fragments andderivatives thereof (such as portions thereof). The nucleotide sequencemay be of genomic or synthetic or recombinant origin, which may bedouble-stranded or single-stranded whether representing the sense oranti-sense strand.

The term “nucleotide sequence” or “nucleic acid” in relation to thepresent invention includes genomic DNA, cDNA, synthetic DNA, and RNA.Preferably it means DNA, more preferably cDNA sequence coding for thepresent invention.

Due to degeneracy in the genetic code, nucleotide sequences may bereadily produced in which the triplet codon usage, for some or all ofthe amino acids encoded by the original nucleotide sequence, has beenchanged thereby producing a nucleotide sequence with low homology to theoriginal nucleotide sequence but which encodes the same, or a variant,amino acid sequence as encoded by the original nucleotide sequence. Forexample, for most amino acids the degeneracy of the genetic code is atthe third position in the triplet codon (wobble position) (for referencesee Stryer, Lubert, Biochemistry, Third Edition, Freeman Press, ISBN0-7167-1920-7) therefore, a nucleotide sequence in which all tripletcodons have been “wobbled” in the third position would be about 66%identical to the original nucleotide sequence however, the amendednucleotide sequence would encode for the same, or a variant, primaryamino acid sequence as the original nucleotide sequence.

Therefore, the present invention further relates to any nucleotidesequence that has alternative triplet codon usage for at least one aminoacid encoding triplet codon, but which encodes the same, or a variant,polypeptide sequence as the polypeptide sequence encoded by the originalnucleotide sequence.

Furthermore, specific organisms typically have a bias as to whichtriplet codons are used to encode amino acids. Preferred codon usagetables are widely available, and can be used to prepare codon optimisedgenes. Such codon optimisation techniques are routinely used to optimiseexpression of transgenes in a heterologous host.

Typically, the nucleotide sequence encoding polypeptides having thespecific properties as defined herein is prepared using recombinant DNAtechniques (i.e. recombinant DNA). However, in an alternative embodimentof the invention, the nucleotide sequence could be synthesised, in wholeor in part, using chemical methods well known in the art (see CaruthersM H et al (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al (1980)Nuc Acids Res Symp Ser 225-232).

Molecular Evolution

Once a promoter-encoding nucleotide sequence has been isolated, or aputative promoter-encoding nucleotide sequence has been identified, itmay be desirable to modify the selected nucleotide sequence, for exampleit may be desirable to mutate the sequence in order to prepare an geneproduct in accordance with the present invention.

Mutations may be introduced using synthetic oligonucleotides. Theseoligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al (Biotechnology (1984)2, p 646-649). Another method of introducing mutations into nucleotidesequences is described in Nelson and Long (Analytical Biochemistry(1989), 180, p 147-151).

Instead of site directed mutagenesis, such as described above, one canintroduce mutations randomly for instance using a commercial kit such asthe GeneMorph PCR mutagenesis kit from Stratagene, or the Diversify PCRrandom mutagenesis kit from Clontech. EP 0 583 265 refers to methods ofoptimising PCR based mutagenesis, which can also be combined with theuse of mutagenic DNA analogues such as those described in EP 0 866 796.

A third method to obtain novel sequences is to fragment non-identicalnucleotide sequences, either by using any number of restriction enzymesor an enzyme such as Dnase I, and reassembling full nucleotide sequencescoding for functional proteins. Alternatively one can use one ormultiple non-identical nucleotide sequences and introduce mutationsduring the reassembly of the full nucleotide sequence. Suitable methodsfor performing ‘shuffling’ can be found in EP0 752 008, EP1 138 763, EP1103 606. Shuffling can also be combined with other forms of DNAmutagenesis as described in U.S. Pat. No. 6,180,406 and WO 01/34835.

Thus, it is possible to produce numerous site directed or randommutations into a nucleotide sequence, either in vivo or in vitro, and tosubsequently screen for improved functionality of the encodedpolypeptide by various means. Using in silico and exo mediatedrecombination methods (see WO 00/58517, U.S. Pat. No. 6,344,328, U.S.Pat. No. 6,361,974), for example, molecular evolution can be performedwhere the variant produced retains very low homology to known proteins.Such variants thereby obtained may have significant structural analogyto known proteins, but have very low amino acid sequence homology.

As a non-limiting example, In addition, mutations or natural variants ofa polynucleotide sequence can be recombined with either the wild type orother mutations or natural variants to produce new variants. Such newvariants can also be screened for improved functionality of the encodedpolypeptide.

Plant Transformation Vectors

Plant transformation vectors of the present invention will contain“expression cassettes” comprising 5′-3′ in the direction oftranscription, a promoter sequence as described in the presentinvention, a gene coding sequence as discussed above and, optionally a3′ untranslated, terminator sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

The promoter sequence may be present in one or more copies, and suchcopies may be identical or variants of the promoter sequence asdescribed above. Such copies may also be complete or partial sequencesas described above.

The terminator sequence may be obtained from plant, bacterial or viralgenes. Suitable terminator sequences are the pea rbcS E9 terminatorsequence, the nos terminator sequence derived from the nopaline synthasegene of Agrobacterium tumefaciens and the 35S terminator sequence fromcauliflower mosaic virus, for example. A person skilled in the art willbe readily aware of other suitable terminator sequences.

The expression cassette may also comprise a gene expression enhancingmechanism to increase the strength of the promoter. An example of suchan enhancer element is that derived from a portion of the promoter ofthe pea plastocyanin gene, and which is the subject of InternationalPatent Application No. WO 97/20056.

These regulatory regions may be derived from the same gene as thepromoter DNA sequence of the present invention or may be derived fromdifferent genes, from Nicotiana tabacum or other organisms, for examplefrom a plant of the family Solanaceae, or from the subfamilyCestroideae. All of the regulatory regions should be capable ofoperating in cells of the tissue to be transformed.

The gene coding sequence may be derived from the same gene as thepromoter DNA sequence of the present invention or may be derived from adifferent gene, from Nicotiana tabacum, or another organism, for examplefrom a plant of the family Solanaceae, or from the subfamilyCestroideae.

The expression cassette may be incorporated into a basic planttransformation vector, such as pBIN 19 Plus, pBI 101, or other suitableplant transformation vectors known in the art. In addition to theexpression cassette, the plant transformation vector will contain suchsequences as are necessary for the transformation process. These mayinclude the Agrobacterium vir genes, one or more T-DNA border sequences,and a selectable marker or other means of identifying transgenic plantcells.

The term “plant transformation vector” means a construct capable of invivo or in vitro expression.

Preferably, the expression vector is incorporated in the genome of theorganism. The term “incorporated” preferably covers stable incorporationinto the genome.

The vectors of the present invention may be transformed into a suitablehost cell as described below to provide for expression of a polypeptidehaving the specific properties as defined herein.

The choice of vector, e.g. plasmid, cosmid, virus or phage vector, willoften depend on the host cell into which it is to be introduced. Thevectors may contain one or more selectable marker genes—such as a genewhich confers antibiotic resistance e.g. ampicillin, kanamycin,chloramphenicol or tetracyclin resistance. Alternatively, the selectionmay be accomplished by co-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect or transform a host cell.

The term “operably associated” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably associated” toa coding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that comprises a nucleic acid, a promoter sequence or a chimaericgene according to the present invention or an expression vector asdescribed above.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a nucleic acid, a promoter sequence or achimaeric gene of the present invention. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal, yeast or plant cells. Preferably, the hostcells are not human cells.

Examples of suitable bacterial host organisms are gram negative bacteriaor gram positive bacteria.

In one embodiment, eukaryotic hosts such as yeasts or other fungi may bepreferred. In general, yeast cells are preferred over fungal cellsbecause they are easier to manipulate.

The use of suitable host cells, such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

The host cell may be a protease deficient or protease minus strain.

The term “transgenic plant” in relation to the present inventionincludes any plant that comprises a nucleic acid, a promoter sequence ora chimaeric gene of the present invention. Preferably the nucleic acid,promoter sequence or chimaeric gene is incorporated in the genome of theplant.

The term “transgenic plant” does not cover native nucleotide codingsequences in their natural environment when they are under the controlof their native promoter which is also in its natural environment.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences discussed herein, or anyderivative, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used as aprobe to identify similar coding sequences in other organisms etc.

The term “variant” as used herein means a protein expressed from anon-endogenous genetic code resulting in one or more amino acidalterations (i.e. amino acid deletions, additions or substitutions) whencompared with the natural or wild-type sequence within the matureprotein sequence.

Plant Transformation

Techniques for transforming plants are well known within the art andinclude Agrobacterium-mediated transformation, for example. The basicprinciple in the construction of genetically modified plants is toinsert genetic information in the plant genome so as to obtain a stablemaintenance of the inserted genetic material. A review of the generaltechniques may be found in articles by Potrykus (Annu Rev Plant PhysiolPlant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-IndustryHi-Tech March/April 1994 17-27).

Typically, in Agrobacterium-mediated transformation a binary vectorcarrying a foreign DNA of interest, i.e. a chimaeric gene, istransferred from an appropriate Agrobacterium strain to a target plantby the co-cultivation of the Agrobacterium with explants from the targetplant. Transformed plant tissue is then regenerated on selection media,which selection media comprises a selectable marker and plant growthhormones. An alternative is the floral dip method (Clough & Bent, 1998)whereby floral buds of an intact plant are brought into contact with asuspension of the Agrobacterium strain containing the chimeric gene, andfollowing seed set, transformed individuals are germinated andidentified by growth on selective media.

Direct infection of plant tissues by Agrobacterium is a simple techniquewhich has been widely employed and which is described in Butcher D. N.et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J. P. Helgeson, 203-208.

Further suitable transformation methods include direct gene transferinto protoplasts using polyethylene glycol or electroporationtechniques, particle bombardment, micro-injection and the use of siliconcarbide fibres for example.

Transforming plants using ballistic transformation, including thesilicon carbide whisker technique are taught in Frame B R, Drayton P R,Bagnaall S V, Lewnau C J, Bullock W P, Wilson H M, Dunwell J M, ThompsonJ A & Wang K (1994). Production of fertile transgenic maize plants bysilicon carbide whisker-mediated transformation is taught in The PlantJournal 6: 941-948) and viral transformation techniques is taught in forexample Meyer P, Heidmann I & Niedenhof I (1992). The use of cassavamosaic virus as a vector system for plants is taught in Gene 110:213-217.

Further teachings on plant transformation may be found in EP-A-0449375.

In a further aspect, the present invention relates to a vector systemwhich carries a nucleotide sequence or construct according to thepresent invention and which is capable of introducing the nucleotidesequence or construct into the genome of an organism, such as a plant.The vector system may comprise one vector, but it may comprise twovectors. In the case of two vectors, the vector system is normallyreferred to as a binary vector system. Binary vector systems aredescribed in further detail in Gynheung An et al., (1980), BinaryVectors, Plant Molecular Biology Manual A3, 1-19.

One extensively employed system for transformation of plant cells usesthe Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid fromAgrobacterium rhizogenes An et al., (1986), Plant Physiol. 81, 301-305and Butcher D. N. et al., (1980), Tissue Culture Methods for PlantPathologists, eds.: D. S. Ingrams and J. P. Helgeson, 203-208. Aftereach introduction method of the desired promoter or construct ornucleotide sequence according to the present invention in the plants,the presence and/or insertion of further DNA sequences may be necessary.If, for example, for the transformation the Ti- or Ri-plasmid of theplant cells is used, at least the right boundary and often however theright and the left boundary of the Ti- and Ri-plasmid T-DNA, as flankingareas of the introduced genes, can be connected. The use of T-DNA forthe transformation of plant cells has been intensively studied and isdescribed in EP-A-120516; Hoekema, in: The Binary Plant Vector SystemOffset-drukkerij Kanters B. B., Alblasserdam, 1985, Chapter V; Fraley,et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985)4:277-284.

Plant cells may be grown and maintained in accordance with well-knowntissue culturing methods such as by culturing the cells in a suitableculture medium supplied with the necessary growth factors such as aminoacids, plant hormones, vitamins, etc.

Culturing and Production

Host cells transformed with the nucleic acid, promoter sequence orchimaeric gene of the present invention may be cultured under conditionsconducive to the growth of the host.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions.

Production of the Gene Product

Expression of a DNA coding sequence or nucleic acid sequence in theplant host cell will produce an RNA transcript. This transcript may be aRNA molecule which is not subsequently translated into a proteinproduct, but which may interact with the gene expression process via themechanism of RNAi, antisense, or cosuppression. If the coding sequenceis derived from a structural gene, the RNA transcript may then betranslated into a protein gene product. If desired, the gene product canbe isolated by standard techniques for isolating proteins frombiological systems, such as salt precipitation, column chromatography,immunoaffinity techniques, electrophoresis, recrystallisation,centrifugation, and such like.

Expression of a Gene Product Detrimental to Plant Cells

If the DNA coding sequence or nucleic acid sequence is translated into aprotein gene product which has a detrimental effect on the plant hostcell, this can be used in a plant cell death system, by using thepromoter of the present invention promoter to direct expression inparticular plant cells. Suitable proteins having a detrimental effectcould include proteases, restriction endonucleases, membrane transportproteins, and ribonucleases such as Barnase. Other examples includeribosome-inactivating proteins (RIPs) such as the maize RIP b-32protein, ricin, abrin, modeccin, barley translation inhibitor andpokeweed antiviral protein (PAP). Pokeweed (Phytolacca americana)produces three distinct antiviral proteins, namely PAP′, PAPII andPAP-S. International Patent Publication No. WO02/33107 demonstrates theuse of truncated variants of the PAP-S protein in a cell death system.Hence, the use of a Ribosome Inactivating Protein (RIP) such as PokeweedAntiviral Protein (PAP) or variants thereof would provide a plant celldeath system.

The invention will now be described, by way of example only, withreference to the following Examples.

EXAMPLE 1 Isolation of the ATC 085 Promoter (SEQ ID No. 7) Region, andProduction of Transformation Constructs

Genomic DNA of tobacco leaf tissue was isolated using the DNAeasy PlantMiniprep Kit (Qiagen) according to the manufacturers' instructions fromK326 tobacco leaf material. A 1 μL aliquot of a 1:10 diluted preparationof genomic DNA was used for a primary PCR using 1 μL of a PCR primerS2PCLOFWD (SEQ ID No. 2) at 10 pM/μL and 1 μL of a PCR primer S2PCLOREV(SEQ ID No. 3) at 10 pM/μL in a 25 μL reaction containing 0.5 μLElongase Taq DNA Polymerase (Gibco BRL), 1 μL Elongase buffer A, 4 μLElongase buffer B, 1 μL dNTPs at 5 mM each, 15.5 μL double distilledwater. The PCR reaction conditions were: an initial cycle at 94° C. for2 minutes followed by 35 cycles of 94° C. for 30 seconds, 60° C. for 30seconds and 68° C. for 4 minutes, followed by a final cycle of 68° C.for 10 minutes. The PCR reaction was electrophoresed on a 1.2% agaroseTBE gel, containing traces of ethidium bromide, at 4V/cm for 2 hours.Two PCR products were seen. The larger product of approximately 1600 bpwas excised from the gel and the DNA purified using the QiaQuick gelextraction kit (Qiagen) following the manufacturers' procedures.

The purified PCR product was cloned into TOPO vector (Invitrogen) andtransformed into TOP10 Chemically Competent E. coli (Invitrogen)following the manufacturers' standard procedure. An aliquot of thereaction was plated onto sterile LB agar containing kanamycin and theplates were incubated overnight at 37° C. Colonies were screened by PCRusing the conditions above to identify clones containing the promoter.The sequence was further confirmed by sequencing.

The ATC085 promoter was excised from the TOPO vector as a HindIII-BamHIfragment and cloned into the plant transformation vector pBin 19 Plusvector based on pGPTV-Kan (Becker et al, 1992) derived from pBIN19(Bevan et al, 1984) in front of three different reporter genes to givethe constructs, GUS (ATC vector pBNP085-0040-001), GFP (ATC vectorpBNP085-0003-001), and Luciferase (ATC vector pBNP085-0017-001). Thepromoter was also cloned in front of an effector gene, the active formof the pokeweed antiviral protein S from Phytolacca americana (ATCvector pBNP085-0501-001). pBNP085-0501-001 has been deposited byAdvanced Technologies (Cambridge) Ltd, 210 Cambridge Science Park,Cambridge CB4 0WA under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the purposes of PatentProcedure at the National Collection of Industrial, Marine and FoodBacteria (NCIMB) 23 St. Machar Street, Aberdeen Scotland, GB on 20 Sep.2005 under accession number NCIMB 41343.

EXAMPLE 2 Isolation of the ATC 023 Promoter Region and Production ofTransformation Construct

The ATC023 promoter was isolated by PCR from Arabidopsis thaliana(Columbia ecotype) genomic DNA using PfuTurbo DNA Polymerase(Stratagene), and the primers AT4G29190L (SEQ. ID. No. 5) and AT4g29190R(SEQ. ID. No. 6) containing restriction sites for HindIII and BamHIrespectively, at an annealing temperature of 63° C. The restrictionsites were then used to clone the isolated fragment into the planttransformation vector pBI101 in front a GUS reporter gene to give theconstruct pBI101 023-0101-001. The construct was then transformed intoE. coli DH5α.

EXAMPLE 3 Production of Transformed Tobacco Plants

The constructs produced above were transferred into the Agrobacteriumtumefaciens strain LBA4404 and transgenic tobacco var. K326 plants wereobtained by in vitro transformation of leaf discs using theco-cultivation method of Horsch et al (1985). Transformed callus andregenerating shoots were selected on MS Agar media containing 100 μg/mlkanamycin and cultured in the presence of Claforan to remove theAgrobacterium cells. Plants were then kept and multiplied in vitro onmedia without Claforan to confirm absence of Agrobacterium.

Plants were transferred to soil in the greenhouse for characterisation.The plants showed growth characteristics indistinguishable from the wildtype tobacco plant in the greenhouse. PCR analysis was conducted toidentify transgenic plants.

For the production of subsequent generations plants can be selfed andseed collected from mature seed heads and stored at 6° C. Segregationratios in progeny from selfed transgenic plants can be used to selectseed batches that contain single insertion sites based on a 3:1 ratio.The number of inserts can also be determined by Southern hybridisation.The insertion into the tobacco genome can be characterised by genomewalking.

EXAMPLE 4 Production of Transgenic Arabidopsis Plants

The constructs described above were transferred into the Agrobacteriumtumefaciens strains LBA4404 and GV3103, and used to transformArabidopsis thaliana by the floral dip method of Clough and Bent (1998).After seed collection from dipped plants, T1 transformants were screenedusing selection on 40 μg/ml kanamycin on ATS/Phytagel™ or 50 μg/mlkanamycin on MS/Agar media. Independent transformants were isolated inthis way and these were transferred to soil and grown under long-dayconditions.

Plants were allowed to self-pollinate to generate T2 seed. Approximately60 T2 seed from the independent lines was screened again on kanamycin,and Chi squared tests were used to determine whether the segregationratio observed was consistent with a single site of transgene insertion(3 resistant:1 sensitive). Multiple independent lines with ratiosconsistent with a single insertion site were identified, and theremaining lines were discarded.

EXAMPLE 5 Characterisation of the ATC 085 Promoter-GUS Expression inTobacco Plants

Sixty plants transformed with the vector pBNP085-0040-001 were used forGUS analysis. After approximately 6-8 weeks growth in the greenhousewhen the apical bud had reached the “floral button” stage, the plantswere either topped using a scalpel at the internode above the 18^(th)leaf (counting from the base), wounded by excising half of the blades ofleaves 16, 17 and 18, or left untreated. After 24 hours, various tissuesamples were taken, including stem sections at the base of lateral buds14, 15, 16, 17 and 18, internodal stem sections, apical flowering shootswhere present, leaf blades and petioles, and primary and secondaryroots. All tissue collected was immediately immersed in 0.1M potassiumphosphate buffer pH 7.0 containing 0.1% β-mercaptoethanol to preventbrowning.

Staining solution was prepared as follows: 300 mg X-gluc powder wasdissolved in 3 ml DMSO in a glass container and made up to 1 L with 0.1Mpotassium phosphate buffer containing 0.5 mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 10 mM EDTA, 0.1% Triton-X and 0.067%Sarcosyl. This solution was stored at 4° C. in darkness.

For staining the tissue samples, the potassiumphosphate/β-mercaptoethanol buffer was replaced with fresh stainingsolution ensuring the tissue was fully submerged, and the samples wereincubated overnight at 37° C. in the dark. The staining solution wasthen replaced with 70% ethanol and the tissues were stored in the darkat room temperature until they had cleared. It was sometimes necessaryto change the ethanol at least once for leaf material. Once the tissuehad cleared, the ethanol was replaced with acidified glycerol. Sampleswere observed and photographed under a microscope.

GUS expression was detected in the plants after all three treatments(topped, wounded, or untreated) and this was localised at the base ofthe lateral buds and/or in the stem close to the point of lateral budinitiation in every case (FIG. 3). A large proportion of the plantsexhibited expression at every bud position sampled. Expression at thelateral buds was detected irrespective of the pre-treatment (topping,wounding, or untreated). In the large majority of plants, no expressionwas detected in any other tissues—only in a few anomalous plants wereany other sites of expression observed, predominantly at the cut edgesof the wounded leaves. Hence the ATC085 promoter was shown to be apromoter directing substantially specific expression at or around thepoint of lateral bud initiation.

EXAMPLE 6 Characterisation of the ATC 085 Promoter-GFP Expression inPlants

Plants transformed with the vector pBNP085-0003-001 were used foranalysis of GFP expression. Whole plants or plant parts, sections ofplants or plant tissues were irradiated with blue light and inspectedfor green fluorescence either by eye or through a microscope.

EXAMPLE 7 Characterisation of ATC 085 Promoter—Luciferase Expression inPlants

Plants transformed with the vector pBNP085-0017-001 were used foranalysis of Luciferase expression. Whole plants, parts of plants orsections of plants or plant tissues were moistened or sprayed with 1 mMluciferin (Sigma) and incubated in the dark for 15 minutes. Luciferaseexpression could then be visualised either by autoradiography onto X-rayfilm, or by photography using a digital camera with a long exposure timeor by using a photon capture device such as an image intensifier linkedto a camera system.

EXAMPLE 8 Characterisation of ATC 085 Promoter—PAP Expression in TobaccoPlants

Twenty-one transgenic and five control untransformed (non-co-cultivatedor NCC) plants were grown in the greenhouse, and after 6 to 8 weeks, theplants were topped using a scalpel at the internode above the 18^(th)leaf to remove the apical inflorescence and uppermost leaves asdescribed above. The outgrowth of lateral shoots from the axils of the16^(th), 17^(th) and 18^(th) leaf was monitored daily for 29 days andscored on the following system of size classes:

0=lateral bud abutting the main stem

1=lateral bud on elongated lateral stem (i.e. 1 internode)

2=lateral shoot with 2 internodes

3=lateral shoot with 3 internodes etc.

From these data the mean outgrowth from the three lateral bud positionson each day was calculated for the transgenic and untransformed plants.These data are plotted in FIG. 4. It is clear that the localisedexpression of the Pokeweed antiviral protein directed by the ATC085promoter has caused a significant delay in the onset of outgrowth oflateral shoots stimulated upon topping and a reduction in the amount ofoutgrowth by Day 29 when compared to the control plants. The effect wasparticularly marked in the two uppermost buds 17 and 18 (see FIG. 4a ).Lateral bud 16 showed less outgrowth (FIG. 4b ) upon topping than buds17 and 18, but this was further reduced by expression of the ATC 085-PAPconstruct.

FIG. 5 shows the reduced outgrowth of lateral shoots in ATC 085-PAPtransgenic plants compared to control plants.

EXAMPLE 9 Characterisation of the ATC 085 Promoter-GUS Expression inArabidopsis Plants

The construct pBNP085-0040-001 was used to transform Arabidopsisthaliana plants. Forty T1 plants were used for GUS analysis. The plantswere grown in soil under long day conditions for 3-4 weeks until afterfloral transition when the primary axis had ‘bolted’ to a length between20-40 mm. The plants were then decapitated by removing the main stem apoint approximately 10 mm from the upper part of the rosette. They werethen allowed to continue growing for 24 hours before being removed fromsoil, washed thoroughly in distilled water and assayed for GUS activity.GUS staining was conducted as described above for the tobacco plants,except that whole plants were used.

GUS expression was detected in the plants by highly intense staining atthe base of the plant at the site of lateral bud formation and in thestem nearby. However, expression was also seen in other tissues inseveral of the plants, including the leaf tissues and veins, petioles,stem, and primary roots.

EXAMPLE 10 Characterisation of the ATC 023 Promoter-GUS Expression inTobacco Plants

Forty-two tobacco plants transformed with the construct pBI101023-0101-001 were grown in the greenhouse and treated as described abovefor GUS analysis.

GUS expression was detected in the lateral buds of 85% of the plantsirrespective of the treatment (topped, wounded, or untreated), and mostof the plants exhibited expression at every bud position sampled. Inmany cases localised expression was also seen in the stem close to thepoint of lateral bud initiation. Expression was also detected in othertissues in some plants, predominantly in the flower tissues, butoccasionally in root, stem, leaf or petioles.

Specific localisation of expression to the lateral buds and the nearbystem was observed in 17% of the plants (FIG. 6). Hence the ATC023promoter can be used alone to provide tissue specific expression. It canalso be used in combination with another lateral bud expressingpromoter, such as ATC 085, to provide complementary expression profileswith an overlapping site of action at the lateral bud tissue.

EXAMPLE 11 Characterisation of the ATC 023 Promoter-GUS Expression inArabidopsis Plants

Five independent transgenic T2 lines carrying single inserts of the023-0101-001 cassette were grown in soil, decapitated and stained forGUS analysis as described above.

GUS expression was observed in the axillary buds and adjacent stem, andfloral tissues (FIG. 7). Expression associated with the smaller buds wasthe strongest, and decreased steadily as the buds developed. Thisindicates that the ATC 023 promoter is associated with lateral budinitiation. Some expression was also detected in other tissues in someplants, including the floral tissues.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope of thepresent invention. Although the present invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin biochemistry and biotechnology or related fields are intended to bewithin the scope of the following claims.

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The invention claimed is:
 1. A chimaeric gene comprising: a promotersequence operably associated with a heterologous nucleic acid sequence,the promoter sequence comprising a sequence selected from the groupconsisting of: SEQ ID NO:4; a sequence having at least 95% identity withSEQ ID NO:4; and a sequence capable of hybridizing to SEQ ID NO:4 understringent hybridization conditions, said stringent hybridizationconditions comprising washing for 2×15 minutes with a 0.1×SSC, 0.5% SDSbuffer at a temperature of 65° C.; and wherein the promoter sequence isoperable to direct expression in at least one of a lateral bud and alateral shoot.
 2. The chimaeric gene according to claim 1, wherein thenucleic acid sequence encodes a cytotoxic molecule.
 3. The chimaericgene according to claim 1, wherein the nucleic acid sequence encodes aribosome inactivating protein (RIP).
 4. The chimaeric gene according toclaim 1, wherein the nucleic acid sequence encodes a pokeweed antiviralprotein.
 5. The chimaeric gene according to claim 4, wherein thepokeweed antiviral protein (PAP) is pokeweed antiviral protein S(PAP-S).
 6. A method of modifying morphology in a plant comprisingintroducing into a plant the chimaeric gene of claim 1 and wherein thenucleic acid sequence encodes a cytotoxic molecule.
 7. The methodaccording to claim 6, wherein the nucleic acid sequence encodes aribosome inactivating protein (RIP).
 8. The method according to claim 6,wherein the nucleic acid sequence encodes a pokeweed antiviral protein.9. The method according to claim 8, wherein the pokeweed antiviralprotein (PAP) is pokeweed antiviral protein S (PAP-S).
 10. The methodaccording to claim 6, wherein an outgrowth of at least one of a lateralbud and a lateral shoot is modified.
 11. The method according to claim10, wherein the outgrowth of at least one of the lateral bud and thelateral shoot is one of reduced, prevented and delayed.
 12. The methodaccording to claim 10, wherein the outgrowth of at least one of thelateral bud and the lateral shoot is modified by one of disrupting themetabolism and causing death of cells involved in lateral buddevelopment.
 13. A method of one of regulating the expression of a genein a plant and modifying the metabolism within a cell of a transgenicplant, comprising introducing into a plant the chimaeric gene of claim 1and wherein the nucleic acid sequence encodes a cytotoxic molecule. 14.A recombinant DNA comprising vector DNA and a promoter sequence operablylinked to a heterologous nucleic acid sequence, wherein the promotersequence is selected from the group consisting of: SEQ ID NO:4; asequence having at least 95% identity with SEQ ID NO:4; and a sequencecapable of hybridizing to SEQ ID NO:4 under stringent hybridizationconditions, said stringent hybridization conditions comprising washingfor 2×15 minutes with a 0.1×SSC, 0.5% SDS buffer at a temperature of 65°C.; and wherein the promoter sequence is operable to direct expressionin at least one of a lateral bud and a lateral shoot.
 15. A plantproduced according to the method of claim
 6. 16. A transgenic plantcomprising the chimaeric gene as claimed in claim
 1. 17. The plantaccording to claim 15, wherein the plant is of the family Solanaceae.18. The plant according to claim 17, wherein the plant is of thesubfamily Cestroideae.
 19. The plant according to claim 18, wherein saidplant is of the genus Nicotiana.
 20. The plant according to claim 19,wherein said plant is Nicotiana tabacum.
 21. The plant according toclaim 15, wherein said plant is selected from the group consisting oftomato, cucumber, Petunia, Dianthus, Picea, Pinus, Eucalyptus, Populus,potato, tobacco, cotton, lettuce, eggplant, melon, squash, pea, canola,soybean, sugar beet sunflower, wheat, barley, rye, rice and maize.
 22. Aplant cell comprising the chimaeric gene as claimed in claim
 1. 23. Aplant cell comprising the recombinant DNA according to claim
 14. 24. Asynthesized nucleic acid comprising a promoter sequence comprising SEQID NO:4, further comprising a nucleic acid sequence encoding a cytotoxicmolecule.
 25. The synthesized nucleic acid of claim 24, wherein thenucleic acid sequence encodes a ribosome inactivating protein (RIP). 26.The synthesized nucleic acid of claim 24, wherein the nucleic acidsequence encodes a pokeweed antiviral protein (PAP).
 27. The synthesizednucleic acid of claim 26, wherein the pokeweed antiviral protein (PAP)is pokeweed antiviral protein S (PAP S).